Abstract

Forkhead box class O 3a (FOXO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Although FoxO3 is a well-known transcription factor involved in diverse biological processes, the role of FoxO3 in cigarette smoke (CS)-induced lung inflammation and injury has not been studied. It is, therefore, hypothesized that deficiency of FoxO3 leads to increased susceptibility to CS-induced lung inflammatory response and airspace enlargement. In this article, we show that the levels of FOXO3 are significantly decreased in lungs of smokers and patients with chronic obstructive pulmonary disease, as well as in lungs of mice exposed to CS. Genetic ablation of FoxO3 led to pulmonary emphysema and exaggerated inflammatory response in lungs of mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted NF-κB DNA-binding ability, leading to inhibition of its activity. Targeted disruption of FoxO3 also resulted in downregulation of antioxidant genes in mouse lungs in response to CS exposure. These results suggest that FoxO3 plays a pivotal role in regulation of lung inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of chronic obstructive pulmonary disease/emphysema.

A, Abundance and localization of FoxO3 in lung alveolar/airway epithelial cells of nonsmokers, smokers and patients with COPD. Red color represents the presence of FoxO3 (indicated with arrow), which was decreased in lungs of smokers and patients with COPD. (-) control = negative control which was stained without primary antibody Alv = alveoli; Aw = airway; E = epithelial cells. Original magnification, × 200. B, Immunostaining scores for FoxO3 per cell-type in alveolar and airway regions of the lung. The assessment of immunostaining intensity was performed semiquantitatively and in a blinded fashion. Immunoblot analysis of FoxO3 in whole tissue lysates extracted from the lung tissue (C) and sputum cells (E) of nonsmokers, smokers, and patients with COPD. After densitometric analysis, the values of FoxO3 in lung tissue (D) and sputum cells (F) were normalized against β-actin (loading control). The relative levels of FoxO3 were significantly decreased in lung tissues of smokers and patients with COPD compared to nonsmokers. G, Whole tissue lysates extracted from the lung tissues of nonsmokers, smokers, and patients with COPD were immunoprecipitated with anti-FoxO3 antibody, and immunoprecipitates were subjected to immunoblot and probed with anti-acetylated lysine antibody. H, Relative intensity of acetylated lysine/FoxO3 represents the increased acetylation of FoxO3 in lungs of smokers. Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared to nonsmokers. +P < 0.05, significant compared with smokers.

A, Immunoblot analysis of FoxO3 in whole tissue lysates extracted from lungs of mouse exposed to CS for 3 days and 4 months. B, After densitometric analysis, the values of FoxO3 were normalized against β-actin (a loading control), respectively. C, Nuclear fraction from lungs of mouse exposed to CS for 3 days and 4 months were immunoprecipitated with anti-FoxO3 antibody, and immunoprecipitates were subjected to immunoblot analysis and probed with anti-acetyl-lysine antibody. IgG was used as an isotype control. D, The relative density of acetylated FoxO3 showed increased acetylation of FoxO3 in CS exposed mice lung at 3 days and 4 months. A representative blot is shown. Data are shown as mean ± SEM (n= 3 to 4 per group). ***P < 0.001, significant compared to air-exposed WT mice.

The levels of MnSOD and catalase were measured by RT-PCR (A) and immunoblot (B) at the indicated time points of CS exposures. CuZnSOD was used as a non-specific target protein of FoxO3, and GAPDH was used for loading controls. After densitometric analysis, the values of MnSOD and catalase were normalized against GAPDH, respectively. Gel pictures shown are representative of at least three separate experiments. Data are shown as mean ± SEM (n= 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared to corresponding air-exposed mice. +++P < 0.001, significant compared with CS-exposed WT mice.